ATP-mediated increase in H+ efflux from retinal Müller cells of the axolotl

Abstract

Previous work has shown that activation of tiger salamander retinal radial glial cells by extracellular ATP induces a pronounced extracellular acidification, which has been proposed to be a potent modulator of neurotransmitter release. This study demonstrates that low micromolar concentrations of extracellular ATP similarly induce significant H⁺ effluxes from Müller cells isolated from the axolotl retina. Müller cells were enzymatically isolated from axolotl retina and H⁺ fluxes were measured from individual cells using self-referencing H⁺-selective microelectrodes. The increased H⁺ efflux from axolotl Müller cells induced by extracellular ATP required activation of metabotropic purinergic receptors and was dependent upon calcium released from internal stores. We further found that the ATP-evoked increase in H⁺ efflux from Müller cells of both tiger salamander and axolotl were sensitive to pharmacological agents known to interrupt calmodulin and protein kinase C (PKC) activity: chlorpromazine (CLP), trifluoperazine (TFP), and W-7 (all calmodulin inhibitors) and chelerythrine, a PKC inhibitor, all attenuated ATP-elicited increases in H⁺ efflux. ATP-initiated H⁺ fluxes of axolotl Müller cells were also significantly reduced by amiloride, suggesting a significant contribution by sodium-hydrogen exchangers (NHEs). In addition, α-cyano-4-hydroxycinnamate (4-cin), a monocarboxylate transport (MCT) inhibitor, also reduced the ATP-induced increase in H⁺ efflux in both axolotl and tiger salamander Müller cells, and when combined with amiloride, abolished ATP-evoked increase in H⁺ efflux. These data suggest that axolotl Müller cells are likely to be an excellent model system to understand the cell-signaling pathways regulating H⁺ release from glia and the role this may play in modulating neuronal signaling.NEW & NOTEWORTHY Glial cells are a key structural part of the tripartite synapse and have been suggested to regulate synaptic transmission, but the regulatory mechanisms remain unclear. We show that extracellular ATP, a potent glial cell activator, induces H⁺ efflux from axolotl retinal Müller (glial) cells through a calcium-dependent pathway that is likely to involve calmodulin, PKC, Na⁺/H⁺ exchange, and monocarboxylate transport, and suggest that such H⁺ release may play a key role in modulating neuronal transmission.

Keywords: calmodulin; glia; monocarboxylate transport; pH; protein kinase C.

Graphical abstract

Figure 1.

Photomicrograph of an isolated Müller cell with a H⁺-selective microelectrode positioned in the “near” position next to the apical end of the cell. Scale bar: 20 μm; double-headed arrow shows the direction of electrode movement as it alternately records the potential established by protons “near” the cell and “far” (30 μm) away.

Figure 2.

Agonists of purinergic P2 receptors induce an increase in H⁺ efflux from Müller cells isolated from axolotl retinae. A: representative recording from an isolated axolotl Müller cell; the bar at the top indicates when ATP was applied to the bath. The asterisk toward the end of the trace denotes the time the H⁺ selective microelectrode was moved 200 µm away from the cell. B: mean ATP-evoked changes in the magnitude of the H⁺ efflux resulting from bath application of increasing concentrations of ATP applied to different Müller cells isolated from the same retinae; n = 6 for 3 µM and 100 µM, n = 5 for all other ATP concentrations, error bars represent ± SEM. C: representative trace from another isolated axolotl Müller cell showing the pronounced increase in H⁺ flux resulting from application of 50 µM ADP. D: mean H⁺ efflux from seven axolotl Müller cells in baseline Ringer’s conditions and in the presence of ADP; ****P < 0.0001.

Figure 3.

Adenosine does not mimic the sustained ATP-evoked increase in H⁺ efflux from isolated axolotl Müller cells. A: representative trace with bars indicating the time during which 100 µM adenosine and 1 µM ATP were added to the bath. B: mean H⁺ efflux from seven axolotl Müller cells in baseline conditions, immediately after adenosine addition to the bath, after 200 s in adenosine, and following ATP addition to the bath; P = 0.062 for baseline compared with early adenosine; P = 0.0019 for late adenosine compared with ATP + adenosine.

Figure 4.

Blockers of P2Y receptors and intracellular calcium release reduce the ATP-evoked increase in H⁺ efflux from isolated axolotl Müller cells. A: representative trace shows an attenuated increase in H⁺ efflux when ATP was added to a bath already containing the P2Y inhibitors PPADS and suramin. B: mean ATP-evoked change in H⁺ efflux from nine control axolotl Müller cells compared with nine separate Müller cells isolated from the same retinae bathed in PPADS and suramin; **P = 0.0015. C: representative trace shows an attenuated increase in H⁺ efflux when ATP was added to the bath of an isolated axolotl Müller cell preincubated for 30 min with the endoplasmic reticulum calcium ATPase inhibitor thapsigargin. D: mean ATP-evoked change in H⁺ efflux from eight control axolotl Müller cells compared with 10 other Müller cells isolated from the same retinae preincubated in thapsigargin; ***P = 0.0002.

Figure 5.

Inhibitors of sodium/hydrogen exchange (NHE) and monocarboxylate transporters (MCT) attenuate the ATP-evoked increase in H⁺ efflux from axolotl Müller cells. A: representative trace shows that the increase in H⁺ efflux induced by ATP was attenuated when amiloride, an NHE inhibitor, was subsequently added to the ATP-containing bath. B: mean H⁺ efflux from ten axolotl Müller cells in baseline conditions, after ATP addition to the bath, and after amiloride with ATP in bath, ****P < 0.0001 for ATP compared with ATP + amiloride. C: representative trace shows an attenuated increase in H⁺ efflux when ATP was added to the bath of an isolated axolotl Müller cell already containing the MCT inhibitor 4-cin. D: mean ATP-evoked change in H⁺ efflux from twelve control axolotl Müller cells compared with 10 distinct Müller cells isolated from the same retinae that were preexposed to 4-cin; ****P < 0.0001. 4-cin, 4-Hydroxycinnamate.

Figure 6.

The calmodulin inhibitor CLP attenuates the ATP-evoked increase in H⁺ efflux from both tiger salamander and axolotl Müller cells. A: representative trace from an isolated tiger salamander Müller cell shows the increase in H⁺ efflux induced by ATP was attenuated when CLP was subsequently added to the ATP-containing bath. B: mean H⁺ efflux from six tiger salamander Müller cells in baseline conditions, after ATP addition to the bath, and after CLP with ATP in bath, ***P = 0.0009 for baseline flux vs. ATP; *P = 0.027 for ATP compared with ATP + CLP. C: representative trace from an isolated axolotl Müller cell shows the increase in H⁺ efflux induced by ATP that was attenuated when CLP was subsequently added to the ATP-containing bath. D: mean H⁺ efflux from six axolotl Müller cells in baseline conditions, after ATP addition to the bath, and after CLP with ATP in bath, ***P = 0.0006 for baseline flux vs. ATP; ***P = 0.0005 for ATP compared with ATP + CLP.

Figure 7.

The protein kinase C inhibitor, chelerythrine (Chel), attenuates the ATP-evoked increase in H⁺ efflux from both tiger salamander and axolotl Müller cells. A: representative trace from an isolated tiger salamander Müller cell shows the increase in H⁺ efflux induced by ATP was attenuated when Chel was subsequently added to the ATP-containing bath. This attenuation was reversed when Chel was removed from the bath and only ATP remained. B: mean H⁺ efflux from seven tiger salamander Müller cells in baseline conditions, after ATP addition to the bath, and after Chel with ATP in bath, ****P < 0.0001 for baseline flux compared with ATP; **P = 0.0047 for ATP compared with ATP + Chel. C: representative trace from an isolated axolotl Müller cell shows the increase in H⁺ efflux induced by ATP was attenuated when Chel was subsequently added to the ATP-containing bath. D: mean H⁺ efflux from seven axolotl Müller cells in baseline conditions, after ATP addition to the bath, and after Chel with ATP in bath, ****P < 0.0001 for baseline flux compared with ATP; ***P = 0.0008 for ATP compared with ATP + Chel.

Figure 8.

Blockers of calmodulin and protein kinase C summatively abolish the ATP-evoked increase in H⁺ efflux from tiger salamander Müller cells. A: representative trace from an isolated tiger salamander Müller cell shows a baseline flux with chlorpromazine (CLP) in the bath. Addition of ATP resulted in a small increase in H⁺ efflux that was reversibly eliminated when chelerythrine (Chel) was subsequently added to the ATP and CLP-containing bath. B: mean H⁺ efflux from eight tiger salamander Müller cells in baseline conditions with CLP, after ATP addition to the CLP-containing bath, and after Chel was added to the ATP and CLP-containing bath, **P = 0.0023 for CLP vs. CLP + ATP; ***P = 0.0002 for CLP + ATP vs. CLP + ATP + Chel; ns P = 0.71 for CLP vs. CLP + ATP + Chel. C: mean ATP-evoked change in H⁺ efflux from six control tiger salamander Müller cells compared with eight distinct Müller cells isolated from the same retinae that had CLP and Chel in the bath; ****P < 0.0001.

Figure 9.

Schematic diagram illustrating the proposed signal transduction pathway by which extracellular ATP induces an increase in H⁺ flux from retinal Müller cells.